Correction to: Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems

In the original publication the Supplementary Material and Fig. 2 are incorrect. The correct version is provided in this correction article. The text HBG2 appearing in the article should be read as HBG1.

To generate gRNA expression plasmid, pBlueScript SK+ vector and the U6 promoter sequence amplified from pLentiCRISPR v2 plasmid (Addgene #52961) via PCR were fused by Gibson Assembly. Annealed oligos for AsCpf1 or LbCpf1 gRNA scaffold were ligated to the pBlueScript-U6 vector to generate gRNA basic plasmids. gRNA expression plasmid for each target site was constructed by ligating the annealed corresponding oligos to the basic plasmid. gRNA oligos were designed through www.Benchling.com. Primers were listed in Table 1.

Cell lines and transfection
HEK293T and U2OS cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin and maintained at 37 °C and 5% CO2. MCF7 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin and maintained at 37 °C and 5% CO2. Transfections were performed in 12-well plates using 200ng of respective dCpf1-p300core expression plasmids and 800ng of equimolar pooled gRNA expression vectors with polyethylenimine (PEI) solution. 48 hr later, genomic DNA was harvested for PAGE and T7E1 assays, or total RNA was harvested for gene expression assay.

T7 endonuclease I (T7E1) assay
PCR amplicons containing target genomic region were purified with Qiaquick PCR Purification Kit (Qiagen). Purified PCR products were denatured and cooled down to form heteroduplex DNA in NEB buffer 2 using a thermocycler. Annealed PCR amplicons were incubated with T7 endonuclease I (NEB) for 90min at 37 °C and subjected to 2% agarose gel electrophoresis.

Western blotting
The HEK293T cells transiently transfected with dCpf1-p300core plasmids were lysed in 2XSDS loading buffer (100 mM Tris pH 6.8, 4% SDS, 20% glycerol, 200 mM β-mercaptoethanol, 0.05% bromophenol blue) and boiled for 10 min. The lysates were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in TBST containing 0.05% Tween 20, and sequencially incubated with the indicated primary antibodies Quantitative real-time PCR was performed using SYBR Premix Ex Taq II (TAKARA) with a Mx3005p Thermal Cycler (Agilent Technologies). Results are expressed as fold change above mock-transfected cells after normalizing to GAPDH expression using the ∆∆Ct method. All qPCR primers are listed in Table 1.

RNA-seq analysis
Total RNA was isolated from transfected HEK293T cells with RNeasy Mini Kit (Qiagen). Briefly, mRNA was enriched and fragmentated. And random hexamers were used for the first strand synthesis, followed by the second strand synthesis.
After cleaned up, double strand cDNA was added with adaptors and amplified by PCR to finanize library construction. After verified using Qubit 3.0, Agilent 2100 Bioanalyzer and quantitative RT-PCR, libraries were pooled and sequenced on an Illumina HiSeq X Ten with 150bp paired-end module.. Reads were aligned to the hg19 transcriptome using Tophat2, and gene expression was quantified using Cufflinks 2.2.1. Differential expression was defined by a Benjamini-Hochberg adjusted p value (q value | FDR) of <0.05 and fold change of >2 or <0.5.